Parshina EA et al. (2022), The Cytoskeletal Protein Zyxin Inhibits Retinoi...

XB-ART-59127

Int J Mol Sci 2022 May 17;2310:. doi: 10.3390/ijms23105627.

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The Cytoskeletal Protein Zyxin Inhibits Retinoic Acid Signaling by Destabilizing the Maternal mRNA of the RXRγ Nuclear Receptor.

Parshina EA , Orlov EE , Zaraisky AG , Martynova NY .

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Zyxin is an LIM-domain-containing protein that regulates the assembly of F-actin filaments in cell contacts. Additionally, as a result of mechanical stress, Zyxin can enter nuclei and regulate gene expression. Previously, we found that Zyxin could affect mRNA stability of the maternally derived stemness factors of Pou5f3 family in Xenopus laevis embryos through binding to Y-box factor1. In the present work, we demonstrate that Zyxin can also affect mRNA stability of the maternally derived retinoid receptor Rxrγ through the same mechanism. Moreover, we confirmed the functional link between Zyxin and Rxrγ-dependent gene expression. As a result, Zyxin appears to play an essential role in the regulation of the retinoic acid signal pathway during early embryonic development. Besides, our research indicates that the mechanism based on the mRNA destabilization by Zyxin may take part in the control of the expression of a fairly wide range of maternal genes.

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Genes referenced: acss2.2 eef1a1 mrc1 odc1 pou5f3 rxrg zyx
GO keywords: retinoic acid binding [+]
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	Figure 1. Downregulation of Zyxin results in an increase in the rxrγ mRNA level in the Xenopus laevis embryos. (a) Analysis by qRT-PCR of rxrγ and rarγ expression in the middle-gastrula stage Xenopus laevis embryos injected with the control and anti-zyxin MO. Here and below the transcript levels of the housekeeping genes odc1 and eef1a1 were used for the data normalization. Error bars indicate standard deviation. (b) Analysis by qRT-PCR of rxrγ expression at bud stage of Danio rerio embryos injected with the control and anti-zyxin splice MO. (c) Analysis by qRT-PCR of the rescue effect on rxrγ expression of zyxin mRNA. (d) Comparative analysis of the rxrγ transcripts levels in Zyxin knocked-down and control embryos at different stages of development. The inset shows at larger scale the data for stages from 10 to 13. The data presented are averaged from at least three independent experiments.
	Figure 2. Zyxin regulates the stability of rxrγ mRNA by preventing formation of the Ybx1/mRNA complex. (a) Overexpression of ybx1 leads to an increase in the rxrγ mRNA level. (b) Suppression of ybx1 mRNA translation by anti-ybx1 MO decreases the rxrγ mRNA level, while co-expression of ybx1 mRNA restores it, even with some overshooting. (c) RIP experiments demonstrate the ability of Ybx1 to bind rxrγ mRNA, whereas Zyxin reduces this ability. Error bars indicate standard deviation. p-value was measured by Student’s t-test. The data presented are averaged from at least three independent experiments.
	Figure 3. Effects of loss- and gain-of-function of Zyxin on γRXRE and RARE cis-regulatory elements in the luciferase reporter assay. (a) Experimental design. (b) Zyxin overexpression does not result in the activation of pGL3-3xRARE reporter in the presence of RA. (c) pGL3-3xRARE reporter activity induced by RA increased in AC with the downregulated zyxin. (d) Zyxin overexpression does not result in the activation of pGL3-2xγRXRE reporter in the presence of bexarotene. (e) pGL3-2xγRXRE reporter activity induced by bexarotene increased in AC with the downregulated Zyxin. (f) pGL3-2xγRXRE reporter activity increased in AC with the overexpressed ybx1 in the presence of bexarotene. Error bars indicate standard deviation. p-value was measured by Student’s t-test. The data presented are averaged from at least three independent experiments.
	Figure S1. Testing of MO specificity and efficiency. (a) Scheme of the morpholino target sites on X. laevis zyxin mRNA. (b) For testing the specificity, MOs were injected into each blastomere of 2-cell X. laevis embryos in different concentrations: 0,3mM (for anti-zyxin and control MO); 0,8mM for anti-zyxin splice MO and 0,8mM for anti-zyxin splice MO with 50 pg/blastomere zyxin mRNA.The injected embryos were collected at the middle gastrula stage and analyzed for amount of Zyxin by Western blotting with anti-Zyxin antibody (Martynova et al., 2008). The data presented are representative of at least three independent experiments.
	Figure S2. Analysis by qRT-PCR of rxrγ expression. Comparative analysis of the rxrγ transcripts levels in zyxin knocked-down and control embryos at different stages of development. This data corresponds to the data presented in Figure 1d. Error bars indicate standard deviation. P-value was measured by Student’s t-test.
	Figure S3. Testing of RARE and RXRE reporters on the AC explants (a) Activation of pGL3-Promoter Vector-3xRARE/6xRARE/12xRARE in response to retinoic acid. (b) pGL3-Promoter Vector-gRXRE reporter activity was compared between experimental samples with rxrg overexpression and control samples. ACs were incubated in solutions with the RXRs agonist bexarotene or all-trans RA.The data presented are representative of at least three independent experiments.
	Figure S4. Effects of loss- and gain-of-function of zyxin on γRXRE and RARE cis-regulatory elements in the luciferase reporter assay in the absence of RA or bexarotene. (a) Zyxin overexpression does not result in the activation of pGL3-3xRARE reporter in the absence of RA. (b) Zyxin knockdown does not result in the activation of pGL3-3xRARE reporter in the absence of RA. (c) Zyxin overexpression does not result in the activation of pGL3-2xγRXRE reporter in the absence of RA. (d) Zyxin knockdown does not result in the activation of pGL3-2xγRXRE reporter in the absence of RA.The data presented are averaged from at least three independent experiments.
	Figure S5. RA and bexarotene do not affect the distribution of Zyxin between the nucleus and the cytoplasm AC explants were incubated in 1x MMR with 10-5M bexarotene (lanes 1-4) or 10-5M RA (lanes 5- 8). Explants were lysed at stage 18 to separate the nuclei from the cytoplasm, and the resulting fractions were analyzed by Western blotting. The data presented are representative of at least three independent experiments.

References [+] :

Allenby, Retinoic acid receptors and retinoid X receptors: interactions with endogenous retinoic acids. 1993, Pubmed